Membrane-type Matrix Metalloproteinase-3 Regulates Neuronal Responsiveness to Myelin through Nogo-66 Receptor 1 Cleavage

Abstract

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositolanchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-offunction approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.Nogo-66 receptor 1 (NgR1) 2 was originally identified as a receptor for myelin-associated inhibitors (MAIs) (1). NgR1 is a glycosylphosphatidylinositol (GPI)-anchored protein that complexes with LINGO and p75 NTR or TAJ/TROY to mediate neurite outgrowth inhibition of neurons (2). Neutralization of NgR1 attenuates the inhibitory effects of MAIs in vitro and promotes regeneration following CNS trauma in vivo (3). Further studies have suggested that NgR1 plays a protective role in Alzheimer disease (4), that NgR1 variants contribute to increased risk of schizophrenia (5), and that NgR1 limits plasticity in the hippocampus (6) and in the visual cortex (7). Understanding the mechanisms that regulate NgR1 will have important implications for CNS plasticity, disease, and regeneration.NgR1 shed from the cell surface is a soluble fragment spanning amino acids 1-358 (8). This fragment is similar to NgREcto (amino acids 1-310), a dominant-negative protein that promotes axonal regeneration in vitro and in vivo by antagonizing MAI signaling (9, 10). The shed NgR1 product can be detected in human cerebral spinal fluid, indicative of a physiological role for this fragment.NgR1 shedding is blocked by the metalloproteinase inhibitor PKF226 -967. Metalloproteinases are a family of zinc-dependent proteases that include ADAM (a disintegrin and metalloproteinases) and secreted or membrane-type matrix metalloproteinases (MT-MMPs) (11). MT1-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16), and MT5-MMP (MMP-24) anchor to the cell surface with a transmembrane domain, whereas MT4-MMP (MMP-17) and MT6-MMP (MMP-25) attach with a GPI anchor. MMPs have been widely studied for their capacity to degrade the extracellular matrix and to promote invasion and migration of tumor cells; however, it is also known that they can cleave a wide range of ligands and receptors...

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